Accessing evidence to inform public health policy: a study to enhance advocacy.

OBJECTIVES: Improving population health often involves policy changes that are the result of complex advocacy efforts. Information exchanges among researchers, advocates, and policymakers is paramount to policy interventions to improve health outcomes. This information may include evidence on what works well for whom and cost-effective strategies to improve outcomes of interest. However, this information is not always readily available or easily communicated. The purposes of this paper are to describe ways advocates seek information for health policy advocacy and to compare advocate demographics. STUDY DESIGN: Cross-sectional telephone survey. METHODS: Seventy-seven state-level advocates were asked about the desirable characteristics of policy-relevant information including methods of obtaining information, what makes it useful, and what sources make evidence most reliable/trustworthy. Responses were explored for the full sample and variety of subsamples (i.e. gender, age, and position on social and fiscal issues). Differences between groups were tested using t-tests and one-way analysis of variance. RESULTS: On average, advocates rated frequency of seeking research information as 4.3 out of five. Overall, advocates rated the Internet as the top source, rated unbiased research and research with relevancy to their organization as the most important characteristics, and considered information from their organization as most reliable/believable. When ratings were examined by subgroup, the two characteristics most important for each question in the total sample (listed above) emerged as most important for nearly all subgroups. CONCLUSIONS: Advocates are a resource to policymakers on health topics in the policy process. This study, among the first of its kind, found that advocates seek research information, but have a need for evidence that is unbiased and relevant to their organizations and report that university-based information is reliable. Researchers and advocates should partner so research is useful in advocating for evidence-based policy change.

Determination of zinc incorporation in the Zn-substituted gallophosphate ZnULM-5 by multiple wavelength anomalous dispersion techniques.

The location of isomorphously substituted zinc over eight crystallographically different gallium sites has been determined in a single-crystal study of the gallophosphate ZnULM-5, Ga((16-x))Zn(x)(PO(4))(14)(HPO(4))(2)(OH)(2)F(7), [H(3)N{CH(2)}(6)NH(3)](4), 6H(2)O, in an 11 wavelength experiment, using data from Station 9.8, SRS Daresbury. The measurement of datasets around the K edges of both Ga and Zn, as well as two reference datasets away from each absorption edge, was utilized to selectively exploit dispersive differences of each metal atom type in turn, which allowed the major sites of Zn incorporation to be identified as the metal 1 and 3 sites, M1 and M3. The preferential substitution of Zn at these sites probably arises because they are located in double four-ring (D4R) building units which can relax to accommodate the incorporation of hetero atoms. As the crystal is non-centrosymmetric, with space group P2(1)2(1)2, it was also possible to use anomalous differences to corroborate the results obtained from the dispersive differences. These results were obtained firstly from difference Fourier maps, calculated using a phase set from the refined structure from data measured at the Zr K edge. Also, refined dispersive and anomalous occupancies, on an absolute scale, could be obtained using the program MLPHARE, allowing estimates for the Zn incorporation of approximately 22 and 18 at. % at the M1 and M3 sites to be obtained. In addition, f' and f'' values for Ga and Zn at each wavelength could be estimated both from MLPHARE results, and by refinement in JANA2006. The fully quantitative determinations of the dispersive and anomalous coefficients for Ga and Zn at each wavelength, as well as metal atom occupancies over the eight metal atom sites made use of the CCP4's MLPHARE program as well as SHELXL and JANA2006. The results by these methods agree closely, and JANA2006 allowed the ready determination of standard uncertainties on the occupancy parameters, which were for M1 and M3, 20.6 (3) and 17.2 (3) at %, respectively.

Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons.

Abeta-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-Abeta and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-Abeta and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees. Quantitative assessment of binding showed that one antibody, ACU-954 was markedly more effective at blocking ADDL binding than other antibodies assessed. ACU-954 was also found to block ADDL binding to hippocampal slice cultures, attenuate the ADDL-induced loss of dendritic spines and detect "natural ADDLs" in human AD tissue. These results demonstrated that antibodies that bind to and block ADDL binding to neurons can be identified, although their efficacy is conformationally specific since it is not readily apparent or predictable based on the core linear epitope or affinity for monomeric Abeta.

ACORN2: new developments of the ACORN concept.

The density-modification procedures incorporated in ACORN, available in the CCP4 package, have proved to be very successful in solving and refining high-resolution crystal structures from very poor starting sets. These can be calculated from a correctly positioned initial fragment containing between 1 and 8% of the scattering power of the total structure. Improvements of ACORN, reported here and incorporated in the program ACORN2, have lowered the size of the fragment required and examples are given of structures solved with only 0.25% of the scattering power in the fragment, which may be a single atom. Applications of ACORN2 to structures with space group P1 have shown the remarkable property that when the starting point is a pair of equal atoms, or even a single atom placed at the origin, the refinement process breaks the centric nature of the initial phases and converges to phases corresponding to one of the two possible enantiomorphs. Examples are given of the application of ACORN2 to the solution and/or refinement of a number of known trial structures and to the refinement of structures when phases are available either from MAD or from a molecular-replacement model.

SPINE workshop on automated X-ray analysis: a progress report.

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.

ACORN: a review.

The ACORN system was originally developed as a means of ab initio solution of protein structures when atomic resolution data were available. The first step is to obtain a starting set of phases, which must be at least slightly better than random. These may be calculated from a fragment of the structure, which can be anything from a single metal atom to a complete molecular-replacement model. A number of standard procedures are available in ACORN to orientate and position such a fragment. The fragment provides initial phases that give the first of a series of maps that are iteratively refined by a dynamic density-modification (DDM) process. Another FFT-based procedure is Sayre-equation refinement (SER), which modifies phases better to satisfy the Sayre equation. With good-quality atomic resolution data, the final outcome of applying DDM and SER is a map similar in appearance to that found from a refined structure, which is readily interpreted by automated procedures. Further development of ACORN now enables structures to be solved with less than atomic resolution data. A critical part of this development is the artificial extension of the data from the observed limit to 1 A resolution. These extended reflections are allocated unit normalized structure amplitudes and then treated in a similar way to observed reflections except that they are down-weighted in the calculation of maps. ACORN maps, especially at low resolution, tend to show C atoms less well, in particular C(alpha) atoms which fall within the first diffraction minimum of their three neighbours. Two new density-modification procedures (DDM1 and DDM2) and a density-enhancement procedure (ENH) have been devised to counter this problem. It is demonstrated that high-quality maps showing individual atoms can be produced with the new ACORN. ACORN has also been demonstrated to be very effective in refining phase sets derived from physical processes such as those using anomalous scattering or isomorphous derivative data. Future work will be directed towards applying ACORN to resolutions down to 2 A.

A modified ACORN to solve protein structures at resolutions of 1.7 A or better.

ACORN has previously been shown to provide an efficient density-modification procedure for the solution of protein structures using diffraction data to better than 1.3 A. The initial phase set could be obtained from a variety of sources such as the position of a heavy atom, a set of scatterers such as S that had been positioned from anomalous dispersion measurements, a fragment or a very low homology model placed from a molecular-replacement search. Several structures solved using the early version of ACORN have been reported in the literature. Here, the effect of applying the original ACORN procedures at lower resolution is reported and new procedures that yield good-quality maps with data sets of resolution down to 1.7 A are described. These new procedures involve the artificial extension of data to atomic resolution and new density-modification processes that develop density at atomic positions that was previously suppressed. The test calculations were aimed firstly towards a proof of principle using a small fragment of a known structure to demonstrate that the procedure could generate correct density and a derived model in initially empty regions of the cell. Further tests addressed the use of more realistic starting models.

The crystal structure of the globular domain of sheep prion protein.

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.

The structure of S100A12 in a hexameric form and its proposed role in receptor signalling.

S100A12 is a member of the S100 subfamily of EF-hand calcium-binding proteins; it has been shown to be one of the ligands of the 'receptor for advanced glycation end products' (RAGE) that belongs to the immunoglobulin superfamily and is involved in diabetes, Alzheimer's disease, inflammation and tumour invasion. The structure of the dimeric form of native S100A12 from human granulocytes in the presence of calcium in space group R3 has previously been reported. Here, the structure of a second crystal form in space group P2(1) (unit-cell parameters a = 53.9, b = 100.5, c = 112.7A, beta = 94.6 degrees) solved at 2.7A resolution by molecular replacement using the R3 structure as a search model is reported. Like most S100 proteins, S100A12 is a dimer. However, in the P2(1) crystal form dimers of S100A12 are arranged in a spherical hexameric assembly with an external diameter of about 55 A stabilized by calcium ions bound between adjacent dimers. The putative target-binding sites of S100A12 are located at the outer surface of the hexamer, making it possible for the hexamer to bind several targets. It is proposed that the S100A12 hexameric assembly might interact with three extracellular domains of the receptor, bringing them together into large trimeric assemblies.

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

A quick solution: ab initio structure determination of a 19 kDa metalloproteinase using ACORN.

A data set from the metalloproteinase deuterolysin was collected at atomic resolution (1.0 A) with synchrotron radiation. The high resolution allowed the structure to be solved with the new direct-methods program ACORN using the coordinates of the Zn atom as a starting point. The phases obtained from ACORN were of sufficient quality to allow automated building to be carried out in ARP/wARP. Minimal manual rebuilding of the model was required and the structure determination was completed using the maximum-likelihood refinement program REFMAC. The whole process, starting from the processed and merged data and ending with a refined model, required less than 6 h of computational time.

Using electron-microscopy images as a model for molecular replacement.

This review addresses the technical problems encountered while using models based on electron microscopy to generate initial phases for crystallographic studies. The test cases used were the gp6 portal protein with 13-fold rotational symmetry and the human hepatitis virus HepB, a viral assembly with T = 4 icosohedral symmetry.

Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis.

The small, DNA-binding protein GerE regulates gene transcription in the terminally differentiated mother-cell compartment during late stages of sporulation in Bacillus subtilis. This versatile transcription factor shares sequence homology with the LuxR/FixJ/UhpA family of activators and modulates the expression of a number of genes, in particular those encoding the components of the coat that surrounds the mature spore. GerE orchestrates the final stages of coat deposition and maturation that lead to a spore with remarkable resistance properties but that must be responsive to low levels of germination signals. As this germination process is largely passive and can occur in the absence of de novo protein synthesis, the correct assembly of germination machinery, including germinant receptors and energy storage compounds, is crucial to the survival of the cell. The crystal structure of GerE has been solved at 2.05 A resolution using multi-wavelength anomalous dispersion techniques and reveals the nature of the GerE dimer. Each monomer comprises four alpha-helices, of which the central pair forms a helix-turn-helix DNA-binding motif. Implications for DNA-binding and the structural organisation of the LuxR/FixJ/UhpA family of transcription activator domains are discussed.

Crystallization of full-length CysB of Klebsiella aerogenes, a LysR-type transcriptional regulator.

CysB is a positive regulator of transcription of genes involved in cysteine biosynthesis in Gram-negative bacteria and belongs to the large family of LysR-type transcriptional regulators. The full-length protein from Klebsiella aerogenes has been crystallized from solutions containing PEG 8000 in the presence and in the absence of the inducer N-acetylserine by the method of vapour diffusion in hanging drops. For the complexed protein different crystal forms appear in the same drops.

Tumor growth and audiometric change in vestibular schwannomas managed conservatively.

OBJECTIVE: To prospectively define the correlation between changes in tumor volume and audiometric function in vestibular schwannomas managed conservatively. STUDY DESIGN: Prospective longitudinal study. METHODS: Twenty-one patients (age range, 15-84 y; mean age, 63.3 y) with newly diagnosed vestibular schwannomas were enrolled between 1994 and 1999 in a protocol at The Ohio State University Hospital (Columbus, OH) to evaluate the correlation between tumor volume and audiometric change during a period of observation. Patients were evaluated yearly by clinical examination, a standardized internal auditory canal magnetic resonance imaging scan with gadolinium contrast for volumetric analysis, and audiometric function testing. Demographic data, historical features, neurofibromatosis type 2 (NF2) status, initial testing results, and serial testing results were recorded. RESULTS: An increase in tumor volume occurred in 14 of the 21 patients (66%). The pattern of volumetric change was found to be extremely variable. Multiple regression analysis revealed significant correlations of changes in tumor volume with changes in pure-tone average and speech discrimination score (P < .0001 and P = .0021, respectively). Change in tumor volume had greater effect on pure-tone average and speech discrimination score in patients initially with class D audiometric function when compared with those initially in class A (P = .0083 and P = .0245, respectively). The presence of NF2 had an independent protective effect against deterioration of the pure-tone average when compared with patients without NF2 (P = .0125). CONCLUSIONS: This study demonstrated a significant correlation between a change in volume and auditory deterioration in vestibular schwannomas being managed with a trial of observation. A given change in tumor volume appeared to have a greater effect on pure-tone average and speech discrimination score as initial auditory classification declined.

The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography.

Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development. Its initiation is under the control of an expanded two-component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single alpha-helical domain comprising six alpha-helices in an unprecedented fold. The structure contains a helix-turn-helix as part of a three alpha-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the sigmaA-activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.

Virtual temporal bone dissection simulation.

We have developed a working prototype system for the virtual simulation of temporal bone dissection. The system offers a paradigm from traditional practices by integrating technological advances to provide a safer and more cost effective way to learn fundamental techniques used in temporal bone surgeries. We present our methods to provide a real-time interactive volumetric system that obviates the need for physical materials in initial training, and provides a more accessible way for residents to practice and to increase exposure to pathological variance. Finally, we discuss ways to extend this work to more advanced resident training, presurgical planning, and surgical documentation.

A flexible and efficient procedure for the solution and phase refinement of protein structures.

An ab initio method is described for solving protein structures for which atomic resolution (better than 1.2 A) data are available. The problem is divided into two stages. Firstly, a substructure composed of a small percentage ( approximately 5%) of the scattering matter of the unit cell is positioned. This is used to generate a starting set of phases that are slightly better than random. Secondly, the full structure is developed from this phase set. The substructure can be a constellation of atoms that scatter anomalously, such as metal or S atoms. Alternatively, a structural fragment such as an idealized alpha-helix or a motif from some distantly related protein can be orientated and sometimes positioned by an extensive molecular-replacement search, checking the correlation coefficient between observed and calculated structure factors for the highest normalized structure-factor amplitudes |E|. The top solutions are further ranked on the correlation coefficient for all E values. The phases generated from such fragments are improved using Patterson superposition maps and Sayre-equation refinement carried out with fast Fourier transforms. Phase refinement is completed using a novel density-modification process referred to as dynamic density modification (DDM). The method is illustrated by the solution of a number of known proteins. It has proved fast and very effective, able in these tests to solve proteins of up to 5000 atoms. The resulting electron-density maps show the major part of the structures at atomic resolution and can readily be interpreted by automated procedures.

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.